Data Sheet
Contents
1. Note This product is covered under CycLex s patents U S Patent No 7 033 778 and No 7256013 European Patent No 1243658 Japanese Patent No 4267043 Canadian Patent No 2392711 1063 103 Terasawaoka Ina Nagano 396 0002 Japan Fax 81 265 76 7618 e mail info cyclex PRODUCED BY Q CycLex Co Ltd cA ucts are supplied for research use only CycLex CircuLex products and y not be resold modified for resale or used to manufacture commercial To inquire about licensing for components products wit CY 1158 Version 1302042. Fluoro Deacetylated Peptide to reference When Fluoro eptide is used fluorescence intensity should increase whenever there is no HDAC activi asample When there is an inhibitory effect on lysylendpeptidase activity even if there is ctiyity in a sample fluorescence intensity should not increase 4 Not only when an inhibitory effect on HDA inhibitory effect on lysylendpeptidase final fluor Fluoro Deacetylated Peptide instead of Fluoro that does not add HDAC8 Although fluofe when Fluoro Deacetylated Peptide is used occurs in a test sample fluorescence i in test chemicals but also when there is an ce intensity will not increase Please use Substrate Peptide and conduct a control experiment e intensity increases even if HDAC is not added nan inhibitory effect on lysly endpeptidase activity ity does not increase For research use only not for use india c or therapeutic procedures C CY 1158 8 Version 130204 p HDACS8 Deacetylase Fluorometric Assay Kit ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Troubleshooting j When chemicals that have an inhibitory effect on lysylendpeptidase are mixed in a HDAC8 fra purified from various cells or the immunoprecipitate using a specific antibody against HD other proteins precise HDAC8 enzyme activity cannot be measured Since the proteasegambi used in the usual protein purification process inhibit lysylendpeptidase activity strongl the
3. For Research Use Only Not for use in diagnostic procedures Example of Test Results Fig 1 Dose dependency of recombinant HDAC8 30min 600 000 pArA See eee eS eae 500 000 400 000 300 000 200 000 F355 F460 counts 100 000 0 5 10 15 20 25 30 35 40 45 GST HDACS conc ug ml Fig 2 Time course of HDAC reaction 1000000 HS ee HPSS Se SS SSS SS ASS SS SS SS SS SS a 40 ug ml amp 20 ug ml B 10 ug ml pO Ere 4 5 ug ml gt aA 800 000 F 2 5 ug ml 1 25 ug ml OR 0 ug ml 600 000 F 400000 ESR ene Ee Hee eee aes eae F355 F460 counts 200000 vp a ia i a a aa Reaction Time min C CY 1158 10 Version 130204 wn HDACS8 Deacetylase Fluorometric Assay Kit Car yCLeXx User s Manual For Research Use Only Not for use in diagnostic procedures Fig 3 Effect of Trichostatin A on HDAC activity One step method 1 000 000 800 000 600 000 400 000 F355 F460 counts 200 000 C CY 1158 11 Version 130204 p HDACS8 Deacetylase Fluorometric Assay Kit ycLex User s Manual For Research Use Only Not for use in diagnostic procedures References Thiagalingam S K H Cheng H J Lee N Mineva A Thiagalingam and J F Ponte 2003 Hi deacetylases unique players in shaping the epigenetic histone code Ann N Y Acad Sci 983 0 2 Yang X J
4. hen there is an ease Please use out the experiment the following Table 2 Not only when an inhibitory effect on HDAC8 is in test chemicals b inhibitory effect on lysylendpeptidase final fluorescence intensity wil Fluoro Deacetylated Peptide instead of Fluoro Substrate Peptide and p of Positive control and Assay control 2 that does not add HI Although fluorescence intensity increases when Fluoro Deacetydate inhibitory effect on lysly endpeptidase activity occurs in a test che fluorescence intensity does not increase Assay reagents Assay control 1 control 2 Positive control 10X Assay buffer 5 ul 5 uL 5 uL 50X Fluoro Deacetylated Peptide 1 1 uL 1 uL 4 10X Inhibitor or equivalent 5 pL 6 Your enzyme sample z dH O 26 5 26 5 uL 31 5 uL 2 X20 diluted Lysylendpeptidase 5 uL 2 5 uL 2 5 uL 1 Following the table above add Reagent of 2 X20 diluted Lysylendpeptidase 2 Incubate for 30 min or desired len at room temperature Ca 25 C 3 Add 50 uL of 6 2X Stop solution to tach well 4 Read fluorescence intensi icrotiter plate fluorometer with excitation at 360 nm and emission at 460 nm C CY 1158 7 Version 130204 K HDACS8 Deacetylase Fluorometric Assay Kit ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Cautions 1 In order to measure the activity of HDAC8 correctly it is necessary to conduct the co exper
5. hematologic malignancies Curr Opin Hematol 9 322 10 Gao L M A Cueto F Asselbergs and tadja 2002 Cloning and functional characterization of HDACI11 a novel member man histone deacetylase family J Biol Chem 277 25748 25755 C CY 1158 12 Version 130204 K HDACS8 Deacetylase Fluorometric Assay Kit yel ex User s Manual For Research Use Only Not for use in diagnostic procedures Related Products CycLex Cellular Histone Acetylation Assay Kit Cat CY 1140 CycLex HDACs Deacetylase Fluorometric Assay Kit Cat CY 1150 CycLex HDACS8 Deacetylase Fluorometric Assay Kit Cat CY 1158 CycLex SIRT1 Sir2 Deacetylase Fluorometric Assay Kit Cat CY 1151 CycLex SIRT2 Deacetylase Fluorometric Assay Kit Cat CY 1152 CycLex SIRT3 Deacetylase Fluorometric Assay Kit Cat CY 1153 CycLex SIRT6 Deacetylase Fluorometric Assay Kit Cat CY 1156 Anti Acetylated Histone p53 K382 Mouse Monoclonal Antibody Cat CY M1 Anti Histone Deacetylase 1 HDAC1 Rabbit Polyclonal Antibody Cat CY P Anti Histone Deacetylase 2 HDAC2 Rabbit Polyclonal Antibody Cat CY P Anti Human SIRT1 Rabbit Polyclonal Antibody Cat CY P1016 NAD Dependent Deacetylase SIRT1 Cat CY E1151 NAD Dependent Deacetylase SIRT2 Cat CY E1152 NAD Dependent Deacetylase SIRT3 Cat CY E1153 NAMPT Nicotinamide Phosphoribosyltransferase Cat CY E125 NMNATI1 Nicotinamide Mononucleotide Adenylyltransferase E1252
6. uL 125 uL 2 L 4 dH O 26 5 uL 265 uL 1325 uL Total 35 uL 350 uL 1750 uL 3500 u HDACS Assay Procedures 1 Assay method Q Assay reagents Test Vehicle Noiizy Inhibitor sample control control 7 HDACS8 reaction buffer 35 uL 35 uL 35 pL 4 10X Inhibitor or equivalent 5 uL Vehicle for Inhibitor 5uL uL 3 10X TSA 5 uL Buffer for your enzyme sample 10 uL 5 X5 diluted recombinant HDAC8 or 10 pL uL _ 10 uL Your enzyme sample 1 Following the above table add Reagent 7 and initiate reaction by adding 10 uL of 5 X5 dilu to each well and mixing thoroughly Incubate at 2 Read fluorescence intensity for 30 to 60 minutes fluorometer with excitation at 340 nm and reaction while the reaction velocity remains d j and 3 or 4 to each well of the microplate Finally X5 diluted recombinant HDAC8 or om temperature Ca 25 C Alternate procedure 1 Following the above table ad initiate reaction by adding 10 u well and mixing thoroughly Inc at appropriate time to stop t t t ach well of the microplate Finally t HDAC8 or your enzyme sample perature Ca 25 C to 2 minute intervals using microtiter plate ission at 440 nm Measure and calculate the rate of your enzyme to each and measure fluorescence intensity in a microplate fluorescence 2 While the reaction rate Qie add 50 uL of 6 2X Stop solution to each well at 380 nm reader
7. with excitation a 3 The differe indicates the and emission at 440 460 nm Note 1 It is ion of each reagents in a reaction mixture as indicated as below Not C CY 1158 measurement is recommended Version scence intensity between Vehicle control and No enzyme control to change the volume of assay reagents and sample as far as it sets up the final 130204 HDACS8 Deacetylase Fluorometric Assay Kit t ycLex User s Manual For Research Use Only Not for use in diagnostic procedures A 2 Assay control 1 When the chemicals that have an inhibitory effect on lysylendpeptidase come to be mixed in HD fraction purified from various cells or the immunoprecipitate using the specific antibody l HDACS or other proteins precise HDAC8 enzyme activity cannot be measured Since the inhibitors used in the usual protein purification process strongly inhibit lysylendpepti please avoid using any protease inhibitors during the process of protein purification If there is such a possibility please carry out the experiment of Positive control control 1 in the following Table using Fluoro Deacetylated Peptide to referene Fluoro Deacetylated Peptide is used fluorescence intensity should increase whenever there is no HDACS8 activity in your enzyme sample When there is an inhibitory effect endpeptidase activity even if there is HDAC 8 activity in a sample fluorescence intensity shoul increase
8. 4 HDACS8 Deacetylase Fluorometric Assay Kit cLex User s Manual For Research Use Only Not for use in diagnostic procedures Quantitative test kit for histone deacetylase activity CycLex HDAC8 Deacetylase _ Assay Kit ays Cat CY 1158 Intended US 0 cssssscsscesceesnivsssasadarcaadacveamienaincas 1 Qy ANOT aT SAES 1 TOA CUO os ieacascOneaiatesilenttaabatotocianeeauabece 2 Principle Of The Assay 3 Materials Provided ccceesecceeesereeeeeeeees 3 Materials Required but not Provided 4 PER AION S sii conescentsansncucaseisuennuatmneianaadineaases 4 Detailed Protocol vscvcisessvercsseccanersssiaseatencansars 5 7 CANONS sesini yasrexsaretectsdeacouetedearsesansaedenaans 8 TroubleshoOotin scsercasenscasecsssesscantensvenvvede 9 Reagent SCADILILY casccisnssssecssevnsaanacesenerscsasinyas 9 Example of Test Results eeeeeeeeeeeeees 10 IRETSICA CES cisarantesisnecastedqundiuanatoteciacasedinatenen Related PROGUCIS vanisnszsarcecsadsasnsccercussunneryensans 1 Intended Use The CycLex Research Product CycLex Deacetylase Fluorometric Assay Kit detects HDAC activity in lysates Primarily the esearch Product CycLex HDAC8 Deacetylase Fluorometric Assay Kit is designed fo r and sensitive evaluation of HDAC inhibitors using recombinant HDACS8 Additionally an Ittred primary cell cell line or tissue homogenate can be assayed for HDAC8 activity wit ex Research Product CycLex HDAC8 Deacetylase Fluorometric Assay
9. Kit after i o ipitation with an appropriate HDAC8 specific antibody Applications for this kit includ 1 Monitoring the purificati ACs including HDAC1 2 3 and 8 2 Screening inhibitors o of HDAC8 3 Detecting the effects armacological agents on HDAC8 ch use only and not for use in diagnostic or therapeutic procedures CY 1158 1 Version 130204 K HDACS8 Deacetylase Fluorometric Assay Kit ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Introduction HDAC proteins are vital regulators of fundamental cellular events including cell cycle progres differentiation and tumorigenesis 1 2 A small molecule inhibitor of HDAC trichostatin A arrests mammalian cells in both G1 and G2 3 4 while overexpression of HDAC1 inga reduces their growth rate by lengthening the duration of G2 and M 5 TSA ind differentiation of mouse erythroleukemia cells and apoptosis of lymphoid and colorectal In addition TSA treatment of cells expressing the PML zinc finger protein derepresses trai and allows cells to differentiate normally 6 With this precedent HDAC inhibitors are being actively explored as potential agents for the treatment of certain forms of cancer 7 9 The human HDACs are organized into three different classes based on their simil east HDAC proteins 1 2 Class I enzymes are ubiquitously expressed and include HDACI1 d 8 which are homologous to the yeast
10. Quantity Required 100 uL assay Dilute the 10X Assay buffer 1 10 with distilled water Since this is the base buffer for the assay prepare 1 vial f1 ssay buffer mixed with 9 ml distilled water and store 10 ml of assay buffer at 4 C 2 X20 diluted Lysylendpeptidase 5 mAU ml Quantity required 2 5 uL assay Dilute the Lysylendpeptidase 1 20 with 1 1 buffer 3 10X TSA 200 uM Quantity required 5 uL assay Dilute the 50X Trichostatin A 1 5 with say buffer 4 10X Inhibitor or equivalent 10X final coficentration Quantity Required 5 uL assay Dilute Inhibitor or equivalent to desired concentration with 1 1X Assay buffer 5 X5 diluted recombinant Quantity Required 10 uL ass Dilute the ee 1 5 with 1 1X Assay buffer Note Use 5 X5 dil ecombinant HDAC8 within the same day they are prepared Qi top solution 1 50 with dH O 6 2X Stop solution Quantity require Dilute the 1 buffer Final 0 25 mAU ml Lysylendpeptidase and 20 uM Fluoro Substrate CY 1158 5 Version 130204 yclex HDACS8 Deacetylase Fluorometric Assay Kit User s Manual For Research Use Only Not for use in diagnostic procedures Component 1 assay 10 assays 50 assays 100 assa 1 M10X Assay buffer 5uL 50 uL 250 uL 500 pL 2 50X Fluoro Substrate Peptide luL 10 uL 50 uL 10 3 2 X20 diluted Lysylendpeptidase 2 5 uL 25
11. RPD3 protein Class II includes HDAC4 5 6 4 9 and 10 which are similar to yeast HDA1 and are expressed in a tissue specific manner The S lass II HDACs including SIRT 1 to 7 require NAD for enzymatic activity It has been reported that HDAC8 is important for the growth of hum distinct inhibition pattern that differs from that of HDACI and 3 whi identity with HDAC8 These findings lead to open the way to the de pent of selective inhibitors of this subtype as potential novel anticancer therapeutics However the conventional method for measuring HDAC acti In order to measure HDAC enzyme activity it is necessary to p substrate First cells have to be labeled metabolically with ad acid to the culture medium Second radioactive acetylated Following the reaction it is necessary to extract an been released from acetylated histone using ethyl a the radioactivity Although a method for measuring the activity of tylase without the use of radioactive substances was reported in recent years owing to the use of fluor t labeled acetylated lysine as a substrate the reaction product must be separated from the intact substrate and the fluorescent intensity measured by i el or cell lines and has a adioactive acetylated histone as a ivityyby adding radioactive acetic as to be purified from the cells radioactive acetyl group which has re the activity of the enzyme based on et reverse phase HPLC As mentioned abov measureme
12. and E Seto 2003 Collaborative spirit of histone deacetylases in regula a structure and gene expression Curr Opin Genet Dev 13 143 153 w Ogryzko V V T H Hirai V R Russanova D A Barbie and B H Howard 1996 Human fibroblast commitment to a senescence like state in response to histone deacetylasesinhibitors is cell cycle dependent Mol Cell Biol 16 5210 5218 4 Wharton W J Savell W D Cress E Seto and W J Pledger 2000 Inhibition itogenesis in Balb c 3T3 cells by trichostatin A Multiple alterations in the induti d activation of cyclin cyclin dependent kinase complexes J Biol Chem 275 33981 3398 nn Bartl S J Taplick G Lagger H Khier K Kuchler and C Seiser histone deacetylase as a growth factor inducible gene Mol Cell dentification of mouse 5033 5043 6 He L Z F Guidez C Tribioli D Peruzzi M Ruthardt A Z P Pandolfi 1998 Distinct interactions of PML RAR alpha and PLZF RAR alpha co frepressors determine differential responses to RA in APL Nat Genet 18 126 135 N Johnstone R W 2002 Histone deacetylase inhibitors el drugs for the treatment of cancer Nat Rev Drug Discov 1 287 299 CrossRef Medlin o0 Kelly W K O A O Connor and P A Marks clinical trials Expert Opin Investig Drugs 11 1695 Histone deacetylase inhibitors from target to 9 Melnick A and J D Licht 2002 Hi acetylases as therapeutic targets in
13. iments for No enzyme control and Inhibitor control at least once in addition to Vehic control as indicated in the above table Although fluorescence intensity increases dips e control when HDAC enzyme activity is in the sample the increase in fluorescence 1 observed in No enzyme control and Inhibitor control 2 In order to estimate the inhibitory effect on HDAC8 activity in the test chemicals co it is necessary to conduct the control experiment of Vehicle control at least once for eyvery experiment and Inhibitor control at least once for the first experiment in addition t sample as indicated in the above table When test chemicals cause an inhibitory effect on activity the level of increase of fluorescence intensity is weakened as compared with Wehicle control The increase in fluorescence intensity is not observed in Inhibitor control w e to be mixed in crude z the specific antibody xt be measured Since the y inhibit lysylendpeptidase otein purification of Assay control using When the chemicals that have an inhibitory effect on lysylendpeptid HDAC8 fraction purified from various cells or the immunoprecipita against HDAC8 or other proteins precise HDAC8 enzyme activi protease inhibitors used in the usual protein purification proce activity please avoid using any protease inhibitors during the p If there is such a possibility please carry out the
14. low 20C 20uLx1 Below 20 C RecombingMMDACR 0x we 100 Stopb utibn 100plx1 Below 20 C In 2 on manual 1 room temp CY 1158 3 Version 130204 HDACS8 Deacetylase Fluorometric Assay Kit c3 ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Materials Required but not Provided Microplate for fluorometer e Microplate reading fluorometer capable of excitation at a wavelength in the range 350 380 an detection of emitted light in the range 440 460 nm Pipettors 2 20 uL 20 200 uL and 200 1000 uL precision pipettors with disposable ti e multi channel pipette e Microplate shaker e Deionized water of the highest quality e 500 or 1000 mL graduated cylinder e Reagent reservoirs Precautions e Please thaw 2 50X Fluoro Substrate Peptide and 50X Fluoro De Peptide at room temperature before use Then thaw the other reagents in ice and use aft the are completely thawed e Please avoid repeated freezing and thawing of the Recombi AC8 in this kit There is a possibility that the enzyme activity may be inactivated Aliquot Land store at 70 C ce a e Please avoid mixing of protease inhibitors such as PMSF or measured HDAC activity in the sample that will be e Do not use kit components beyond the indicated ki te e Rinse all detergent residue from glassware e Use deionized water of the highest quality e Do not mix reagents from different kit
15. nt systems are difficult to adapt for processing many samples under a variety of s because of their complicated operation Thus a simple system for biochemical analysis or inhibitor screening without the use of radioactive substances is preferred C CY 1158 2 Version 130204 K HDACS8 Deacetylase Fluorometric Assay Kit ye ex User s Manual For Research Use Only Not for use in diagnostic procedures Principle of the Assay CycLex HDAC8 Deacetylase Fluorometric Assay Kit measures the activity of HDAC by the principle of changing an HDAC reaction into the activity of the protease Since it is very simple measure common protease activity and it can be performed at a low price the measuremen This new method of measurement should dramatically raise the efficiency of inhibi biochemical analysis of these enzymes Measuring Principle of The CycLex HDACS8 Deacetylase Fluoro X X X Lys Ac MCA 4 lt Deacetylase X X X Lys MCA t Lysly endpepti X X X Lys AMC Measurement of fluoresce e Note This measuring principle and kit are covere der CycLex s patents U S Patent No 7 033 778 and No 7256013 European Patent No 1243658 Japanese Patent No 4267043 Q Canadian Patent No 2392711 Materials Provided v Each kit contains FQ Quantity Storage Co Iml x2 Below 20 C 100pLx1 Below 20 C fyl ted Peptide 1 mM _ 50uLx1 Below 20 C Lysylendpeplaace AodmAUml Sul x1 Be
16. s e Do not mouth pipette or ingest any of th reagents e Do not smoke eat or drink whe handled a orming the assay or in areas where samples or reagents are Biological samples may be wounds or breathe aero inated with infectious agents Do not ingest expose to open protective gloves and dispose of biological samples properly C CY 1158 4 Version 130204 K HDACS8 Deacetylase Fluorometric Assay Kit ycLex User s Manual For Research Use Only Not for use in diagnostic procedures Detailed Protocol Description of assay system CycLex HDAC8 Deacetylase Fluorometric Assay Kit can measure the enzyme activit with a homogeneous method In this method the reaction is initiated and the fluoresce measured by mixing simultaneously fluorescence labeled acetylated peptide which is subs and lysly endpeptidase Since the reaction is not stopped it is necessary to measure intensity at regular intervals after the reaction is initiated and to determine reaction Velocity Alternatively within a time in which the reaction velocity is kept constant it is also ible to stop the reaction by adding the Stop solution and to measure fluorescence intensity Preparation Method for Assay Reagents Thaw 2 50X Fluoro Substrate Peptide and 50X Fluoro Deacetylated i room temperature Stand other reagents in ice to thaw Use them after they thaw completely 1 1X Assay buffer 20 mM Tris HCl pH 8 0 125 mM NaCl 1
17. use of any protease inhibitors during the protein purification process 2 Final fluorescence intensity will not increase both when test chemicals have an inhibitory HDAC8 and also when there is an inhibitory effect on lysylendpeptidase 3 If the test reagents themselves emit fluorescence at excitation wavelength 80 nm and fluorescence wavelength 440 460 nm the inhibitory effect of the test a a be evaluated correctly 4 The recombinant HDAC8 should be run in duplicate using the prot cribed in the Detailed Protocol Incubation times or temperatures significantly different f e specified may give erroneous results 5 The reaction curve is nearly a straight line if the kinetics of the the first order Variations in the protocol can lead to non linearity of the curve as can as ineti s that are other than first order For a non linear curve point to point or quadratic curve fit should be used ns in the Detailed Protocol were el pipettor maintenance e 6 Poor duplicates indicate inaccurate dispensing instru followed accurately such results indicate a need ti All of the reagents included in the CycLex ch Product HDAC Assay Kit have been tested for stability Reagents should not be used beyo tated expiration date Upon receipt store the Recombinant HDACS at 70 C all other s should be stored below 20 C C CY 1158 9 Version 130204 K HDACS8 Deacetylase Fluorometric Assay Kit f ycLex User s Manual
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